DESCRIPTION (appended verbatim from investigator's abstract): Apoptosis is an active form of cellular suicide that ensures that superfluous or undesirable cells are eliminated. Besides its importance in embryogenesis and physiology, this phenomenon has considerable significance in the etiology and treatment of disorders such as cancer, AIDS and Alzheimer's Disease. The intracellular pathways of apoptosis are not fully understood. However, the basic machinery is largely conserved among species and cell types. We have been studying the mechanisms of apoptosis using a cell-free system based on extracts from eggs of the frog, Xenopus laevis. This system displays authentic biochemical and morphological features of apoptosis, and has been used to uncover novel aspects of the apoptotic process, particularly the function of Bcl-2 in blocking the release of cytochrome c from mitochondria, thereby preventing caspase activation and apoptosis. Recent studies have shown that certain pro-apoptotic relatives of Bcl-2, including Bax and Bid, can interact directly with mitochondria to cause the release of cytochrome c. The research proposed here aims to determine the mechanisms through which Bid and Bax cause the outer mitochondrial membrane to be permeable to proteins like cytochrome c. First, we will determine whether Bax- and Bid-induced cytochrome c release can occur without compromising other aspects of mitochondrial function, particularly protein import via the classical pathway. Second, we will elucidate the molecular mechanisms through which Bax and Bid induce membrane permeabilization, using systems that range in complexity from isolated whole mitochondria down to synthetic liposomes reconstituted with purified or recombinant proteins. Finally, we will use biochemical and genetic methods to identify novel proteins that mediate or modulate the mitochondrial events in Bax- or Bid-dependent pathways. Such studies will help address the question of whether mitochondrial events in the apoptotic program represent a point of no return, or instead can be reversed under certain conditions, resulting in cell survival.